We have developed a primary mammary epithelial cell culture system using collagen gel as a substratum wherein mammary cells proliferate and differentiate into secretory cells producing milk proteins in response to insulin, cortisol and prolactin. Using this system, we showed that epidermal growth factor (EGF), which is formed and secreted by the salivary gland, stimulates mammary cell proliferation and inhibits expression of milk protein genes. These effects of EGF were observed at physiological concentrations. Mammary epithelial cells have been shown to possess specific EGF receptors with a high (Kd = 1 x 10tothe-10M) and a low Kd = 3.6 x 10tothe-9M) affinity. The number of high- and low-affinity receptors was about 800 per cell and 8,600 per cell, respectively. The occupancy of EGF receptors for a half-maximal stimulation of DNA synthesis or inhibition of casein synthesis was about 10% and 6% of total receptors, respectively. These findings implicate possible regulatory role of EGF in the development of the mammary gland. In addition, a potent tumor promoter, 12-0-tetradecanoylphorbol 13-acetate (TPA), was found to simulate the effect of EGF on DNA synthesis and milk protein production in this system. Both EGF and TPA decreased the specific binding of prolactin to mammary cells, and their effects were synergistic. The ability of various TPA analogs to stimulate DNA synthesis and inhibit casein synthesis correlated with their potency as tumor promoters. These results indicate that tumor promoters switch the developmental path of mammary epithelium from differentiation to growth. Cortisol was previously shown to exert concentration-dependent, different effects on the accumulation of casein and alpha-lactalbumin in organ culture of the mammary gland. In the present study, we showed that 30 nM cortisol, in combination with insulin and prolactin, stimulated maximally the synthesis of alpha-lactalbumin and its mRNA accumulation, whereas 3000 nM cortisol decreased the extent of both of these events. On the other hand, casein production was maximally enhanced by 3000 nM cortisol through stimulation of the synthesis and its mRNA accumulation as well as reduced turnover. The inhibitory effect of cortisol on the phenotypic expression of the alpha-lactalbumin gene was selectively reversed by prostaglandines.